1 1 1 1 1
VITAMIN D AND PAROTID GLAND FUNCTION IN THE RAT
1
by
1 1
Charles G. Peterfy
1 1 1
1 1 1 !
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfil1ment of the requirements for the degree of Doctor of Philosophy
October 1988
Department of Pharmacology and Therapeutics McGi1l University Montreal, Canada
1 1 1 1
Copyright
~\
Charles G. Peterfy, 1988
1
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1
1
1
Sorne of the work of this thesis has been pub1ished:
Peterfy, C.
& Tenenhouse, A. (1982). Vitamin D receptors in
iso1ated rat parotid gland acinar ce11s. Biochlm.
Ac ta. 721: 1'58 -163 .
Glijer,
B,
Peterfy,
C. & Tenenhouse,
vivo
Peterfy,
C. T'enehouse,
A., Yu,
E.
(1988).
gland function in the rat. J. Physiol.
1
1 1 1
,
(1985).
The effect of
J. PhyS10l. 363:323-334.
1
1
A.
vi tarnin D deficiency on secretion by rat parotid gland in
1
1 J
Biophys.
i
Vitamin D and parotid
398:1-13.
1 1
ABSTRACT
1 1
This
is a target organ for vi tamin to
1
1
,
characterize
1
, 1 1
o.
Employing methods previously used
1,25-dihydroxyvitamin DJ (l,25(OH)2DJ) receptors
in established target tissues, classical receptors for this sterol were demonstrated
in acinar cells of
Submandibular gland,
lacrimal
normal
gland and pancreatic
1
stimulated and auriculotemporal
acinar
cells
nerve - stimulated parotid saliva,
which persisted when serum concentrations of calcium, parathyroid hormonf' and 1,25(OH)2DJ were maintained within normal limits, yet was
reversed by treatment with vitamin DJ (0 3 ). The concentration
of calcium in pilocarpine - stimulated saliva did not correlate with decreased salivary flow, serum concentration of l"e l ease by Thes€'
exocytosis
findings
but appeared to vary with changes in the this
ion.
Amylase secretion
were normal
suggested
that
in
fluid
and calcium
vitamin D-deprived secretion but
125(011)203 was
not
the active metabolite
for
rats.
not prote:in
secretion by parotid gland was vitamin D dependent. but
that
this effect.
25-hydroxyvitamin DJ
(25011D 3 ), 24,25-dihydrL)xyvitamin D3 (24,25(OH)2DJ) and 1,25(OH)2D3 ta
correct
abnormal parotid function
in vitamin D-deprived rats
revealed that 24, 25(OH) 2D3 was the active metabolite,
J 1 1
gland.
decrease in the rate of production of pilocarpine-
EXdmilldtion of the relative abilities of DJ'
t
rat parotid
did not contain 1,25 (OH) 2D) receptors. Vi tamin D deprivation caused a
1 t 1
thesis examines the hypothesis that the parotid gland
ii
and essen-
1
1 1 1 1 1
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, 1 ,
tial for normal water and electrolyte secretion by parotid gland. Carbachol-stl.mulated potassium vitamin D-deprived rats action of
24,z5(OH)2D3
secretion.
efflux
was normal, was
later
parotid
suggesting tha!' in
the
glands
from
the site of
sequence of
fluid
Sucrose density gradient analysis of sterol binding
in parotid cytosol demonstrated that 24, 25(OH) 2D3 di.d not utlliz(' the
same
reeeptor as
1,25(OH)2D3'
A
specifie
receptor
24,25(OH)2D3 was not clearly identified; however,
for
a role for
cellular Gc -protein in the mechanism of action of 24,25 (OH) 2D3 wns postulated.
The
function was not for
importance of 1,25(OH)2D3 determined,
this rnetabolite in
acinar
al though cèlls
role.
1
1 1 1
1 1
1 1 1
from
iii
in parotid
the presence
suggested that
gland
of receptors i t had
somp
1 1
RESUME
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Lors de ce travaU de savoir
si la
vi tamine D. afin de D3
glande
thèse nous nous sommes intéressés à
parotide était un organe
pour la
En employant les mêmes méthodes que celles utilisét:,:,
caractériser les récepteurs
(l,25(OH)2 D3)
dans
de récepteu:s
niveau des cellules acini de Au contraire,
de la 1, 25-dihydroxyvitamine
des organes cibles connus,
demontrer l'existence
le~
cible
nous
avons pu
classiques pour ce stérol au
la glande parotide de rats normaux.
les glandes submandibullaire et lacrymal ainsi que
cellules acini du pancréas ne contiennent pas
pour la l,Î')(OH)2D3
En réponse à la pilocarpine ou â la stimulâ-
t ion ne rveuse
auriculotf!mporale,
provoque,
l~
chez
de récepteurs
rat,
salive parotidienne.
une
une carence
diminution du
taux de
en vi tamine
D
production de
De plus, cette sécretion anormale de salive
parotidienne persiste même lorsque les cOl'lcentrations sériques de calcium,
d'hormone parathyroidienne
tenups dans des
et de 1,25(OH)2D3 sont main-
1 imi tes normales, mais est renversée lorsque les
ra t~ reçurent une diète contenant de la vitamine 03 (° 3 ).
Lors de
la stimulation salivaire dûe à la pilocarpine, la concentr "ltion de calcium n'est pas en corrélation avec la diminution du fhlX salivaire mais apparaît plutôt changer avec les variations de concentration sérique de cet ion.
D3 plus, la sécretion d'amylase et
la libera tion de calcium par exocytose sont normales chez des rats soumis
~
une
déficience en
vitamine D.
L'ensemble
de ces
résultats suggerent que la sécretion en eau et en électrolytes de
iv
1
1 1
la glande parotide est dépendante de la vitamine D. alors que la
1
sécretion protélque
ne
l'est pas.
De
1 1 1 1 1
la relative habilité de la vitamine D3' de
plus.
la l,25(OH)2D3
n'appaL"ait pas être le metabolite actif de cet effet.
L'étude de
la 25-hydroxyvitamine
D, (250HD 3 ), de la 24,25-dihydroxyvitarnine D3 (24,25(OH)2D3) et de la l,25(OH)2D3 à corriger la fonction anormale de la glande parotide, chez des
rats privés de vitamine D,
montre que
24,25(OH)2D3 est le métabolite actif et qu'il est,
IR
d'autre part,
essentiel pour
la sécretion normale
cette glande.
Le flux sortant de potassium de glandes parotides
d'eau
et d'électrolytes par
stimulés par le carbachol est normal chez des rats privés de vitamine D, suggérant que le site d'action de la 24,25(OH)2D3 est
1
plus
tardif
1
électrolytes.
1 1
sucrose,
1
1 1
1 1 1
dans
cytoplasme des
la
L'analyse
de
sécretion
de la liaison
glandes parotides,
montre que
récepteur que
séquence
en
des stérols
après gradients
1., 24,25(OH)2D3 n'utilise
la l,25(OH)2D3.
Un
eau
et
en
dans le
de densi té de pas
récepteur spécifique
le même pour
la
24,25 (OH) 2D3 n'a pas été identifié avec prée ision mais, toutefois, un rôle
de la
l, 25(OH) 2D3 dans
pr0téine Gc la
est postulé.
fonction de la
glande
L'importance de 1 a parotide n'a
PlI
étre
déterminée bien que la présence de récepteurs pour ce métabolite dans les cellules acini tend à montrer que rôle.
v
ce composé y joue un
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TABLE OF CONTENTS
TABLE OF CONTENTS
vi
LIST OF FIGURES
xi
AKNOWLEDGEMENTS
xv
LIST OF ABREVIATIONS
xviii
PART ONE: INTRO.JUCTION
1.1
Statement of the Prob1em
2
l 2. Chemistry and Metabolism of Vi tamin D ................ .
4
1 3. Regulation of Vi tamin D3 Metabolism . . . . . . . . . . . . . . . . . . .
6
1 3.1. D3 Production ................................. .
6
1.3.2
7
25-Hydroxylation .............................. .
1.3.3. 10: -Hydroxy lation
8
1.3.4. 24-Hydroxylation
10
Classical Actions of Vitamin D . . . . . . . . . . . . . . . . . . . . . .
10
1 4.1. Ca1cwm and Phosphate Homeostasis ............. .
10
1.4.2
Intestinal Actions ............................ .
II
l .4.3. Renal Effects ................................. .
12
1.4.4. Effects on Bone .............................. ..
13
1 5. Non-Classical Effects of Vitamin D . . . . . . . . . . . . . . . . . . . .
15
l . 5 1. Modulation of Endocrine Secretion ............. .
15
1.5.2. Cellular M.qturaLion/DifferentiaCion ........... .
17
1.5.3. Effects in Ske1eCbl Muscle .................... .
19
1 6. Cellular Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . .
20
l .6.1. Genomlc ....................................... .
20
1.6.2. Non-Genomic ................................... .
23
vi
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r
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1.7. Parotid Gland Anatomy and Sa1ivary Composition ....... .
27
1. 8. G1andular Mechanisms of Secret ion .................... .
33
1.8.1.
Class~cal
Two-St:age Theory
................... .
1.8.2. Saliv. ry Calcium Secret:i:m Secret~oll
33
37 ................... .
37
1.8.4. Vascular Element:s in the Salivary Reflex ...... .
39
1.8.5. Hotor Elements in the SalJ.vary Reflex .... .
40
1.8.3. Neural Control of
1 8 6
Cooperat:~vlty
in Secret:ion .. , ................. .
40
1 9. Cellular Mechanisms of Water and Electrolyte Secretion
............................... .
1 .9 1
Na +/K +IC 1- CC'transport. . . . . . ..
1.9.2.
Cr
. ....
and K+ Channel Activation
,
1.9.3. Sources of Activator Calcwm . . . . . . . . . . . . . . . . . .
1 1 1
1.9.5. Identity of the ActIvaCor Pool of Calcium . .... .
50
1.9.6. CalcIum Entry ................................. .
50
, 1
1.9.4. Transduction of Receptor-Hediat.ed Calcium Hobillzacion ............................. .
1.10. Ce llular Mechanisms of Prote in Sf'cre t ion ............ .
1
1
53
1.10.l. The InosltoljPK-C Pathway . . . . . . . . . . . . . . . . . . . .
1.10.2. The cAl1PjPK-A Pathway ....... 1.10 3. CalclUm and the cAMP Pathway
1.10.4
.. . . . . . . . . . . . .. .. . ....... .
57
Interactions Betwaen che InositoljPK-C and cA!1P IPK-A pathways ...... .
l.11. Effec ts of Cale i tropic Hormones .................... .
1 1
49
vii
60
6]
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PART T\
2S-hydroxylase
uv
ultraviolet
1 ]
1 1
1 1
olet
xx
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PART ONE: INTRODUCTION
1 1
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1.1. Statement of the Problem
The importance of vitamin D in bone and mineraI metabolism is weIl established, and it is
generally accepted that its mineralotropic
effects are produced by modulating transepithelial calcium trdnsport in intc-c,tine,
bone and kidney
At
the time l began work on the present
thasis there was a growing awareness that the vitamin D system comprised
]
n more complex scheme th an was originally appreciated.
Identification of
additional target tissues that differed markedly in their oveIall function&,
)
and demonstrations
seemingly unrelated, importance
that vitamin D influenced numerous,
fundamental cellular processes
of vi tamin D extended far
beyond
affirmed that
the
i ts classically reggrded
raIe in mineraI metabolism. Indeed, Braidman et al (1985) have suggested
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1
that regulation of calcium homeostasis by vitamin D actually developed only as an evolutionary afterthought. There existed at t:'1e time no unifying hypothesis to explain the behaviour of vitamin D in such a diversity of cell types,
was generally presurned that sorne aspect of cellular calcium metabolism Iv,]!'.
J 1
1 1 1 1
although it
probably involved, and it hau been speculated that vitamin D played
a regulatory role process.
~herever
calcium translocation was an important
Many exocrine glands transport calcium in large amounts.
Indximdlly stimulated rat parotid gland for example, secretin~.
in l
h
an arnount equivalent
to almost 10%
The
is capable of of the calcium
pl'C'!',C'nt in blood (calculated from the data of Schneyer et al, 1978). The prC'c ise mechanism certain.
of calcium secretion by the
The findings of Wallach et al
(1971),
pr.trotid gland
is
that 80% of the total
cell ular calcium in parotid gland is localized in zymogen granules, that
calcium secretion by
this
tissue
2
not
and
is tightly coupled to pre.tein
1
secretion (later confirmed by Kanagasuntheram et al, 1981), suggestpd R model in which calcium was taken up from tne intersti tial fluid
1 1 1 J 1 1
al
0110
end of the acinar cell, transported in secretory grrulules to the apical surface
at
the
other
secretory proteins essence,
end,
and
finally
and other granular
released
contents
in conjunction wi th
during
exocytosis.
In
this represented a transcellular calcium transport process in
the parotid gland,
and raised the possibility that. vi tarnin D playpd a
role in the normal function of this and perhaps other exocrine glandi->. In support of this hypothesis was the dFmonstration by Goodwin
al (1978) of vitarnin
~t
that rat parotid gland accumulat2d the principal metabolitf'b D,
and contained a calci.um binding pn)tein simi1ar to the
III
vitarnin D- dependent,
1 1
1 1 1 1 1 1
1 1 1 1
et al D3
ca1ciurn- binding protein descrihed
tn kidney
Aoki
(1979) reported that a binding protein for 1,2, - dihydroxyvi t1lJuin
(l,25(OH)2 D3)
metabolite
similar
to
the
intracellular
rect::ptor
for
this
in classical target organs was present in homogenates frolll
rat parotid gland.
Christakos et al
(1981) also reported evidence for
l,25(OH)2D3 receptors and calcium-binding protein in this tissue. Takiguchi
et: al
(1980) had shown that
vitamin D modulated microsoma1
Ca 2 + -ATPase in rat parotid gland, and Tenenhouse et al (1978,
1979) had
demonstrated an effect of vitamin D on the rate of protein secretioll by parotid gland using vitamin D-deprived and glucocorticoid-treated rat5. Considering the relationship between protein s,'cretion and transce11ula)" calcium transport suggested by the
experim~r~s
of Wallach et al (1971).
this latter finding was particularly compelling. Predicated upon these findings and speculations.
the experimpnt..yvitamin D3
the C-24 position on the side chain (250HD)"24-
24,2S-dihydroxyvitamin
(250HD 3 ,
D3
24, 25(OH) 203 and
regarded as the principal metabolites of metabolites
4
°3 ,
(24,25(OH)2D3)' l, 25 (OH) 2D3)
These
are generally
However, at least 16 other
1
are a1so formed under in vivo conditions (Norman et al,
1982), and th"
question of what physiologiea1 significance each of these rnay have has
.1
1 J
1 1 1 1
1 t 1 1 1 i 1 1
1 1
,
been a subject of live1y controversy since the mid-1970' s. Prevailing views bio10gieal aetivity
maintain that
only
1,25 (OH)2D3
possesses
at physiologieal coneentrat!.ons (Brornrnage
1985). 1,25(OH)2D3 has a much shorter p1a.::ma
half-l~fe
any
et al,
(0.5-3 h: GHyet
al, 1978; Fraser et al, 1986; Lor, 1986) than either 250HD 3 (1-2 weeks: Clements et al,
1987) or 24,25(OH)2D3 (3 days: Jarnagin et al,
1985),
and is regu1ated within a narrow concentration range severa1 fold
low~r
than that of 250HD 3 or 24,25(OH)2D3' A1though a11 three metabolites have demonstrab1e aetivity in assays of elassical vitamin D-dependent actionb in bone and intestine, 1,25 (OH) 2D3 is the most potent and rapid1y acting (Halloran et
al,
deprived animaIs,
1981a;
Stern,
1981).
In ncphrectomized,
which 1ack 1Q-OHase ae tivity
exclusively in rena1 proximal tubules (Kawashima
vitarnin D-
(lQ-OHase is
located
et al,
1983»,
1981,
l, 25 (OH) 2D3 is the only metabolite that stimu1ates intestinal absorption of calcium, and bone resorption (Boyle et al, 1972). Moreover,
animaIs
given l,25(OH)2D3 as the sole source of vitamin D do not have any identifiable abnormalities (Jarnagin et al, Several
groups
have
neverthe1ess
1983). sugges ted tha t
metabolites of vitamin D may have actions distinct 1,25 (OH) 2D3' 24,25(OH)2D3'
Particular whieh
is,
from those of
attention in this regard has under
normal
add i t 10na 1
eircumstances,
foeused on the
major
dihydroxylated form of vitamin D in the circulation. Desp i te a number of proposed actions, upon for
however,
2!~,25(OH)2D3
no physiologieal ro1e
has yet been
agrE'(~d
or any other analogl.le of D3 besides 1,25(0l1)?D 3
(see Brornrnage et al, 1985). If 24,25(OH)2D3 has no function, then the question of the r01e of
5
1 1 1
1 1 1 1
24-0Hase must be addressed.
1 1 1
1 ~
~
i
, , 1 1
interesting in
this regard that la-
hydroxylation is the only metabolic tran&formation of vitamin D in which the A- ring is further modified. Ali others,
including 24 -hydroxylation
represent sorne form of alteration of the 8-carbon side-chain of vitamiu D. The principal mechanism of inactivation of 1,25(OH)2D3 is by cleavage of this side chain (re"iewed in Norman et: al, 1982, and Kurnar,
1984),
and this process is initiated by 24-hydroxy1ation
1986).
24-0Hase appears (Tanaka et al.
al,
to
he present in
1974; Kumar et: al,
1985; Reiche1 et: al,
l,25(OH)2D3 (Chandler et al, of these observations
1
It is
(Horst et al,
a11 target tissues
of 1,25 (OH)2D3
1978; Howard et: al, 1981; Gamhlin et:
1986),
and its synthesis
is induced by
1984). The most widely held Interpretation
is that 24-0Hase serves sole1y as
an inactivator
of the vitamin D system: Ini tiating degradation of 1,25 (OH) 2D3 at sites of 1,25 (OH)2D3 action and synthesis, as weIl as diverting the metabolism of 250HD 3 to 24, 25(OH) 2D3'
1.3. Regulat'lon of Vitamin D3 Metabo1ism
1. J .1. DJ P!'oduct:ion. Photochemical synthesis of D3 from epiderma1 7 -dehydrocholesterol nonenzymatic event.
in response
to
ultraviolet
(UV)
radiation
is
a
The principal determinants of production rate are
net UV exposure and
7-dehydrocho1estero1 concentration. Decreasing
epidermal stores of 7 - dhydrocholesterol with advancing age (Adams et al, 1982)
may contribute
elder1y
(Gallagher et al,
7-dehydrocholesterol however, three
in part
to
abnorma1
1979 ;
calcium metaholism
in the
Tsai et al, 1984). Large stores of
present in the skin under normal conditions
provide a potentially excessive
minimal erythemal exposures
6
capaci ty for
D3 production:
of the total human body
to UV
1 1 1 .1
,)
1
1 1
1 1
1 1 1
1 1 1
1 1 1
irradiation can raise serwn concentrations of D3 10-fold (Holick et al, 1981) . Recent evidence suggests that sorne degree of indirect regu1ation of this process rnay nevertheless occur. stirnu1ate the
synthesis of me1anin
l, 25(OH) 2D3 has been shawn ta
(Hosoi
et sl,
1985),
and
promot€'
terminal differentiation of epidermal cells, and thus the formation of cornified skin (Hosomi et al, 1983; see also Holick et al, Collectively,
.::hese effects
1987).
of 1,25 (OH)2D3 would lirnit the penetrllLi on
of UV light, and thus indirectly Inhibit the production of 03'
1.3.2. 25-Hydroxy1ation. 25-hydroxylation of D3 occ·.us exclusively in the
liver.
Cytochrome
P 450-dependent
25-0Hase
systems have
been
identified in both mi tochondrial and micrasornal fractions of rat liver (Bhattacharyya et al, 1974; Bjo't'khern et al, hydroxy lation
has generally
been regarded
1978). Microsomal 25-
to be
important (Fraser, 1980, Kumar, 1986); although, Dahlback et al
(1987)
in rats
que~tion
this,
physiologica1ly morE:' recent experiments by
and Saarem et al
(1984)
report that on1y a mitochondrial 25-0Hase is present in hwnan liver. Regulation of 25-hydroxylation has not been exarnined extensively probab1y because until quite recently, 25-0Hase was not believed to bp major
site of regulation
supported
by
the
of vitamin D metabo1isrn.
observa t ion
that
concentrations in subj ects receiving normal
levels
250H0 3
II
This view was
accumula tes
to
h igh
large doses of vitamin D3' dcc.,pj te
of 1,25(OH)2D3' HowE:ver, vitarnin D-deficient subjectc; do
convert vitamin 03 to 250HD 3 better than vitarnin D-replete subjects, and the high concentration of 250H0 3 found in osteoporotic wornen deCreab(;!5 during treatment with 1,25(OH)203 (Lor, 1986). Moreover,
inhibition of
25 -OHase by 1,25 (OH) 2D3 has been demonstrated in rat liver (Baran et al,
7
1
1
1983).
changes may be due to al tered 250HD 3 clearance (see also: Bell et al, 1987; Clements et al, 1987).
1
1 1 1 1 1
1 1 1 1 1 1
1.3.3.
1
1
1 1
1cr-Hydroxylation.
250HD 3 -lcr-OHase
is
a
cytochrome
P450 -
dependent mono-oKygenase present in the non-pregnant rat eKclusive1y in mitochondria of
the renal proximal
tubule
(Kawshima et
al,
1981;
Kawashima et al, 1983). Although this is genera11y be1ieved to represent the
normal
localization of lex-OHase,
limi ted sensitivi ty of existing
i t must be noted that due to the
assay methods,
lcr-OHase activity can
on1y be detected under conditions favouring enzyme stimulation (Warner et
1985).
al,
deprivation.
Experimentally this is
usually achieved by
vitamin
D
The assertion that la-OHase has a similar distribution in
normal, vitamin D-replete rats can therefore only be assumed.
~xtrarena1
1ex-hydroxy1ation clear1y does occur: in placenta during pregnancy (Pike et
1979;
al,
Tanaka et
al,
1979;
Zerwekh
et al,
1986),
and
in
pathological conùitions such as sarcoidosis (Barbour et al, 1981; Mason eta1,1984).
1a-OHase activity is influenced by a number of agents. important of these
is parathyroid hormone (PTH).
through which decreased e1evation 1977; Cl-.
1
Thus, regulation of 25-0Hase may occur, although many of these
i st S
of
circulating
Kawashima et al, sOlne
is the mediator
plasma calcium concentration resu1ts 1,25(OH)2D3
concentration
1981; see Audran et al,
(Horiuchi
et
in al,
1985). A1though there
difference of opinion on the matter, evidence suggests that
1,25(OH)2D3
feedback inhibits
(Russell
al,
et
PTH
The most
1986;
Silver
the synthesis and secretion of PTH et al,
1986).
Withdrawal
of tonic
inhibi tian of the parathyroid glands by l, 25(OH) 2D3 has been implicated in the pathophysiology of secondary hyperparathyroidism in renal failure
8
1 1
1 1 J J
1 1 1
1 1 1
1 1
1 1 1
1 1
(Korkor, 1987; Sherwood,
1987). The effect of PTH on 1a-OHase activity
appears to be mediated by cyclic AMP (reviewed in Kawashima et al, 1986)
via
a rapid
(minutes) mechanism
(Rasmussen et al,
(Henry,
1979)
Rost et 81,
(Larkins et al, 1974), and a
1981) requiring no new prote in synthesis slow mechanism (hours)
1972;
that is dependent on prote-in
synthesis (see Kawashima et al, 1986). Decreased plasma phosphate has a direct stimu1atory effect on 1aOHase (Rasmussen et al, 1972; Hughes et al, 1975; Gray et al, 1985). The mechanism is not weIl understood, growth hormone status.
but appears to be dependent on normal
l, 25(OH}2D3 feedback inhibits
la-OHase
(Henry,
1979). Other less established regulators include pro1ac tin (Robinson et
al, 1982) and growth hormone (Spanos et al, 1978, Spencer et al, 1981). Although the
effects of calcitonin are
controversia1,
evidenc('
suggests that it selectively stimulates la-OHase present in the- proximal straight tubules (PST) of the kidney via a non-cAMP-mediated mechanism (Kawashima et al,
1981).
action of PTH on
la-OHase in proximal
This contrasts with
the known cAMP-depende>nt
convoluted tubules
(PCT),
and
suggests that two anatomically and functionally distinct 1a-Olias(' systems coexist in the nephron (Kawashima et al, sens i tive
1a-OHase wou1d
be expected
to be
latent
deprivation due ta the associated hypocalcemia; stated at
the beginning of this
section,
1986). A calcitoninduring
and yet,
vitand n
D
for rf'élS011',
it was precise1y under such
conditions that arguments supporting the exclusive rena1 loca1ization of 1a-OHase were developed.
It is
therefore possible
ta
specu1ate
that
under conditio!ls favouring higher calcitonin leve1s additiona1 sites of 1,25(OH)2D3 synthesis, although undetectab1e by present technology, emerge.
9
mlly
1 t 1
1 ]
1.3.4. 24-Hydroxylation. 24- and lcr-hyroxylation are in many ways similar processes
(reviewed in Kawashima
1986);
there
is sorne
speculation that the 24-0Hase is a cytochrome P450 system like lcr-OHase. (Tanaka et al.
Despi te the fact tha'':: 24-0Hase is found in many tissues 1974;
et al,
Kumar
1978;
Reichel et al, 1986),
it
et al,
Howard
has not
regulation is not weIl understood.
j
et al,
a~
Gamb1in et al, 1985;
1981;
yet been isolated,
and its
In general, 24-0Hase activity appears
to vary inversely with that of lcr-OHase. As previously mentioned, 1,25(OH)2D3 activates 24-0Hase. 24-hydroxylation in turn initiates the
i J
1 1
1
inactivation
of 1,25(OH)2D3 (Horst et
processes form a homeostatic feedback 1,25(OH)2D3
concentration.
PTH,
a
activi ty and 1, 25(OH) 2D3 production,
al,
1986).
loop
principal
Together these
in the
regulation of
stimulator of
b10cks the
lcr-OHase
l, 25(OH) 2D3-stimu1ated
activation of 24-0Hase via a cAMP-mediated mechanism at doses 10wer than those required to induce lcr-OHase (Shigematsu et al, 1986; Lor,
1986).
A more thorough examination of this enzyme and its regulation is needed.
.1 1.4. Classical Actions of Vitamin D
;1
.)
1 .4.1.
Calcium and Phosphate
examined effects of vitamin D are
Homeos tasis.
The
most
thoroughly
those involved in the regulation of
calcium and phosphate homeostasis (reviewed in Audran et al, 1985, and
:1
J
Fraser
et al,
parathyroid glands, secret ion of
1 1 1
1986). Briefly, hypoca1cemia is first
PTH.
which immediately (minutes) PTH causes a
rapid
results
sensed by the in increased
increase in plasma calcium
concentration by lowering plasma phosphate concentration, and thus the
10
~------l
1
1 1
1 J
1 1 1
1 1
1
calcium-phosphate product (Ca 2+xp) via a direct phosphaturic effect on the kidney.
PTH a1so decreases renal calcium clearance.
and mobilizes
calcium stores in bone by stimulating osteoc1astic resorption. A direct stimulatory effect of PTH on intestinal calcium transpc.rt has re-cpnt1y been disc10sed by Nemere et al (1986). As mentioned above. PTH inhibits 24-0:-1ase and stimulates la-OHase
activity,
thus rE>orienting renal
metabolism of 250HD 3 toward l,25(OH)2D3 production. PTH also indtnctly stimulates
la:-OHase
concentrations. Thus, of
l,2~(OH)2D3
rise.
activity
by
lowering
plasma
phosphate
in a matter of hours, circu1ating concentrations l,25(OH)2D3
enhances intestinal calcium and
phosphorus absorption, and decreases renal clearance of bath calc ium and phosphate.
1,25 (OH) 2D3 itself stimulates osteoclastic bone l'esorption,
but also augments PTH-mediated osteolysis. As plasma calcium concentration rises in response ta the concerted
effects of PTH and l, 25(OH) 2D3' it
feedback inhibits secretion of PTH.
Elevated calcium also stimulates release of ca1citonin,
which direclly
inhibits osteoclastic bone resorption, and may a1so oppose altered renal calcium/phosphate handling.
Furthermore,
1,25 (OH) 2D3
i tself
feedback
In pure hypophosphatemia lo-0Hase is activated directly,
but PTJI
1
inhibi ts PTH secretion.
1
secretion is not stimu1ated since lowering the Ca 2+xP product tends to
1
1 J
1
1 1
raise plasma intestinal
calcium concentration.
phosphate
absorption
ta
This
allows
l,25(OH)2D3-dependpnt
proceed wi thout
associated
PTH-
mediated phosphaturia.
1 .4.2. appears
to
Intestinal Act:ions. comprise
both
Calcium
saturable
absorption by
and unsaturable
the
intestine
components,
transcellu1ar as weIl as paracellular routes, and utilize both active
Il
1
1 1
1 1
1 1 J
1 1 1 1 1 1
1 ,1 1
1 1
transport and simple diffusion. The subjec· has been reviewed in detall by Wasserman (1981) and Wasserman et al (1985). Breif1y,
transcellular
calcium flux ineludes an initial uptake across the brushborder membrane at
the luminal
surface
transfer through finaUy three
of the
enterocyte,
followed by intracellular
the cytoplasmic compartmen't to the serosal pole,
extrusion of components,
calcium which
across
the baso1ateral membrane.
implicitly
involve
differir,g
and
These
molecular
processes, prE-sent the enterocyte wi th different phys iologieal p"'oblems. Several models have been proposed to integrate these features of calcium transport
taking into account the problAm of the potential toxici ty of
calcium t:> the cell. Calcium uptake across the brushborder membrane is generally believed to be a passive process.
Once inside,
ealciUlT' must
associate with intracellular organelles or binding proteins way
as
to
nontoxic capacity
maintain its
levels.
Although mitoehondria
for aecumu1atiol' of
ini tially considered for
(Nemere,
1986;
this role,
Nemere,
(in
view of
cytoplasm at their
calcium) and Golgi reeent work has
activity of 4S+Ca 2+
highest specifie lysosomes
ionized concentrations in
in sueh a low,
recognized
apparatus were revealed that
(cpm/mg protein) is
the
actually
in
Leathers et al, 1986). The vitamin D-
dependent, cytosolic calcium-binding protein, calbindin-D, has al 50 been implicated
in
such
a
proteetive
raIe,
a1though
this
remains
controvers ia1 (see be1ow: 1.6. Cellular l1echanisms of Action).
1.4.3.
Renal Effects.
Primari1y due
establishing controlled experimental conditions,
to
diffieulties
in
studies examining the
effects of vitamin D on rena1 function have yielded conflicting results (reviewed in Kawashima et al,
1986, and Kumar,
1986). Wel1 controlled
investigations (Pusehett et al, 1972; Liang et al, 1982; Brezis et al,
12
1 J 1 1 1 1 1 1
Fetal and
neonata'
vitamin O-deficient
mineralization and growth until Mathews
al,
eL
1984).
after
rats
weaning
The development
receptors,
(Halloran et al,
1981b).
essential for skeletal minera1ization.
,
in these
1,25 (OH) 2°3
Thus,
l,25(OH)203
cannot
be
There is sorne evidence, how('ver,
that 24,25(OH)203 is important in this process (Tam et al, 1986). Widening of osteoid seams in bone is a classicai rickets and osteoma1atia (Geoffrey et al, 1986) phosphate
concentrations are sirnu1taneously
deficient
rats,
resorbed.
This
the excess osteoid is resu1ts
with
in an
al though,
1,25 (OH) 203
care
administration to
1 1 1 1
1983;
al,
changes
intes tina1
skeleta1
and emergence of vi tamin 0- dependent calcium and phosphorus
absorption
Treatment
1
(Miller et
of rachitic
animaIs correlates wi th the appearancE' of
increase
the
sterol
must
be
restores taken
in
correc ted in 1,25 (011) 2D3-
trabecular bone
normal
with
(net resorption
of
When plasma calcium and
but
not
volume
and
Weinstein et al,
1984;
lnsure physiological
in plasma
feature
norma11y mineralized,
t.otal bone ash weight (Underwood et al,
1
exhibit normal
trabecular bone
the
dosage
and
1984).
turnover; method
steady-state concentrations of bone
resu1ts if
of of
circulating
1,25 (OH) 2D3 rises on1y slightly above normal (Gallagher et al, 1986». Thus,
the
specifie action of
1,25 (OH) 203
stimulation of bone resorption,
which
on bone appears
to
bl?
is necessary not on1y for
t}w
mobi1ization of bone mineraIs during calcium/phosphate homeostasis,
but
also for normal ske1etal rnode11ing and remode11ing. Exactly how
1,25 (OH) 2D3 promotes
bone
resorption is
not c1Û8r.
Both the number and activity of osteoclasts are increased by 1.25(OH)2D3 (Holtrop et: al, 1981; Hefley et al, 1982). Osteoclasts are believed to
1
derive from b100d monocytes,
1
resorption
1
(Ash et al,
1980;
which also have a Bonucci,
14
1 ~--------
1981;
ca~aclty
for honp
Sminia et al,
1986).
1 1 )
Circulating monocytes possess intracellular receptors for l, 25 (OH) 2D3' and differentiate preferentially toward the macrophage/osteoclast phenotype in response to thp sterol (Bhalia et al, 1983; Provvedini et Ill,
of
]
1 J 1
1983; Suda et al, 1986; also see below: 1.5. Non-Classical Effects ~'itamin
D).
Osteoclasts however,
1ack receptors
for 1,25(OH)2D3
(Marke et al, 1986); activation of these celis by l,25(OH)2D3
via lymphokines, such as osteoc Iast - ac tivating
therefore i ndirec t factor,
which are
is
secreted under monocytic control (Domingut.....,: et al,
1979; Yoneda et al, 1979). Osteoclasts also lack receptors for PTH, and stimulation by this peptide appears to
be
indirect
via sorne
as
yet
undefined intermediate cell (Perry, 1986).
J
1 )
1.5. Non-Classical Effects of Vitamin D
Numerous addi tional effects of vitamin D have been described which do not appear to
J J
1 1 )
1 1 1 1
be directly concerned with
the maintainance of
calcium/phosphate homeostasis or ske1etal integrity.
1.5.1.
Modulation of Endocrine Secretion.
possess receptors for l,25(OH)2D3'
and havE' been shown te alter their
secretory activity in response to changes "secondary"
talget
tissues
are
postulated
branch of the classical vitamin D system, in calciumjphospha te homeostas is
Many endocrine glands
or
in vitamin D status. to
comprise an
These
auxiliary
and to play an accessory role the regulation of vi tamin
D
metabolism. In this model,
the
inhibitory effec.: of 1,25(OH)2D3
on the
secretion of PTH, the principal inducer of renal la-OHase, 15 viewed as
15
1 1
1 1
a feedback mechanism by which 1,25(OH)2D3 (Russell et al,
1986;
Silver et al,
1 1 1 1
1 1 1 1
1 1 f
1
Escape of the parathyroid di~appearance
of
sterol receptors and deve10pment of tissue insensitivity is suspected to under ly secondary
hyperparathyro idism
comp1 icating
rena1
fallur€'
(Korkor, 1987; Sherwood, 1987).
pancreas CB-cells) 1975),
hava
(Clark et al,
also been demonstrated 1980),
pituitary (HaussIer et al,
medu11a (Clark et al,
1986),
in endocrine
parathyroid (Brumbaugh et 81,
1980;
Stumpf et al,
testis (Levy et al,
1985;
1982),
adn'nal
Stumpf
1987), ovary (Dokoh et al, 1981) and placenta (Christakos et al, and 1,25(OH)2D3 has
been reported
to alter the
(Norman et al, 1980) and pro1actin (Wark et contr01led experiments (Tanaka et al, secondary changes deprivation Moreover,
in
plasma calcium
secretion of
al,
fOt
81,
1980), insulin
1982). Carefully
1986) however, suggest that
concentration
and treatment may account
during vi tamin
for many of
normal TRH-stimu1ated PRL secretion,
D
these effects.
GH responses
and HCG-
stimu1ated testosterone secretion were observed in patients with vitamin D-dependent
rickets
type-II (VDDR II)
hereditary disease characterized by tissue
(Hochberg et
81,1985),
a
insensltivity to 1,25(OH)2D3
due to abnorma1 1,25(OH)2D3 receptors (Brooks et al, 1978; Balsan et al, 1983; Hirst et al, 1985). The few patients that exhibited a weak insul1n response to
glucose.
and decreased TRH-stimulated TSH
also found to be severely hypocalcemic.
secretion wprc
In these subjects,
normal
secretion was restored by infusion of calcium. Apart from the effects on para thyroid gland,
the bulk of evidence does not support a direc
regulatory role for 1, 25(OH)2D3 in endocrine secretion.
1
,
1986).
its own biosynthesis
gland from tonie inhibition by 1,25 (OH) 2D3 due t0 the
l, 25(OH) 2D3 receptors
1
limits
16
t
1 1 1 1 1 1 l 1 1 1
1 ]
j
1.5.2. Cellular Haturat::ionjDifferent::iation.
intriguing non- c 1assical action of vi tamin D is i ts trophic effect on ce 11 growth,
di vision and differentiation.
Promotion of terminal
differentiation of skin by 1,25(OH)2D3 has already been mentioned (Hosomi et al, insights
1983; Holick et al,
have
corne
from
1987),
but the most compe11ing
investigations
of
J J 1 1
the
rhe importance of vitamin D in hematopoiesis was first recognized by
Uris t
and
McLean
in 1956:
they
noted pronounced
proliferation in the marrow of hypocalcemic, Anemia
and
extramedu11ary hematopoiesis
mast
ce11
vitamin D-deficient rats.
are
known
complications
of
vitamin D-dependent rickets in children, and respond to treatment with vitamin D (Yetgin et al, 1982). Recurrent infection and impaired immune response also complicate rickets and osteomalacia (Stroder, peripheral macrophages
from
vitamin D-deficient
1975),
and
children exhibit
defertive motility and phagocytosis (Stroder et al, 1970; Lorente et al, 1976). Similar abnorma1ities of macrophage function in vitamin Ddeprived mi ce are corrected by 1,25(OH)2D3 in vitro
(Bar-Shavit et al,
1981). Normal circulating monocytes possess 1,25 (OH) 2D3 receptors, preferentially
al,
1983;
receptors
j
immunity and
hemato1ymphopoietic system.
differentiate
to macrophages
and possibly
lmder the influence of this sterol (Bhalla et:: al,
1
Probably the most
Suda
et al,
(Manolagas et
1986).
al,
express the 1a-OHase enzyme therefore
(Adams
and under
et al,
osteoclasts
1983; Provvedini et
AU macrophages a1so have
1985),
and
cert~in
1983);
1,25(OH)2D3
circurnsances,
it i5 specu1ated
that l, 25(OH) 2D3 may play an autocrine ro1e in these cells
(Rook et
al,
1987). ThE" presence of 1a-OHase in macrophages may a1so
underly
extrarena1
l, 25(OH) 2D3 production
17
and
hypercalcemia
in
1 )
granulomatous diseases sueh as sarcoiJ.osis (Barbour et 81, et al, 1983; Mason et al, 1984).
1
1 1
1 1 1
1 1 1 1 1 1
Normal resting B- and T-lymphocytes do
not contain receptors for l,25(OI-l)2D3 (Bhulla et al, 1983; ProvvE'dini f.'t
al,
1 l,
1983);
(mitogens,
reCE!ptors appear however, Epstein-Barr virus)
(Bhulla et al, Moreover,
1
once
1983; Provvedini et al,
1,25 (OH) 2D3
these (,, loOHD 3 > 24,25(OH)2D3) (Weeks1er et al, 1980); moreover, the rank order of reeeptor affinities for the different sterol metabolites corre1ates bioassays
with
(Stern,
nuclei with high temperature (Pike,
their relative 1981).
in classica1 vitamin
1,25(OH)2D3 receptors
but extremely
D
also bind to purified
affinity (subnanomolar K d ).
independent,
1982).
potencies
This
sensitive to
interaction is ionie disruption
End- organ insensitivity to 1,25 (OH)2D3 in patients wi th
VDDR I I in which receptors bind the sterol normally, but laek affini ty for ehromatin (Hirst et al, 1985), attests to the importance of nuclear binding. The intraeel1ular localization of unoecupied 1,25(OH)2D3 receptors
1
in vivo was initially believed to be cytosolic like that of classieal steroid receptors. 1,25 (OH) 2D3 More
In eOlltrast to steroid receptors however,
receptors required
If
stabilization"
reeent experimet'\ts have demonstrated that
receptors aetually
in hypertonie
cytoso1ie buffers.
unoecupied 1,25 (OH) 2D3
eopurify with the nue1ear fraction if buffer ionie
strength is deereased;
in extremely hypotonie media,
the proportion of
total 1,25(OH)2D3 reeeptors bound to nuclei approaches 100% (Walters et
al,
1
1
1980).
Recent
immunocytoehemieal studies also support
21
a
1 1
1 .1
1 1 1 1
predominant1y nue lear localiza tion of
the unoccupied 1,25 (OH) 2 D3
receptor (see Pike, 1985). Binding of 1,25(OH)2D3 inereases the receptor's affinity for ehromatin in terms of the critical ionic strength required to dissociate them
(Hunziker
al,
et"
"transformati on".
1983).
This presumab1y reflects
receptor
Based on biochemical di fferences between s terol-
receptor complexes genereted in vit"ro and in vivo, Hunziker et al (1983) propose
that
s tep proces s
1,25(OH)2D3-receptor
transformation is probably a
multi-
as pos tulated for glucoeorti.co id- receptor ae t i vat ion
(Bailly et al, 1980). Al though defini tive evidenee
is lacking,
transformed l, 25 (OH) 2D3
reeeptors are believed to alter transcription by interacting with specifie
regu1atory
sequences
in
1,25(OH)2D3-sensitive
genes
1
1985).
l,25(OH)2D3
et al,
1974), and enhances intestinal chromatin temp1ate activity
1 1 1 1
(Zerwekh et al, 1976). These effects are dependent on establishment of
1
1 J
1 1
1
the
stimulates DNA-dependent RNA-polymerase II
(Pike,
l,25(OH)2D3-rec.eptor
comp1ex,
and
u1timately
production of new proteins which presumab1y
(Zprwekh
result
.nediate
the
in
the
cellular
effects. Post-trans1ationa1 modulation of prote in synthesis via effect.s on mRNA processing has b8en suggested (Bronner et al,
1982; Dupret et
al, 1986). Two specifie vitamin D-dependent proteins,
al,
1984),
and the
ealeium-binding protein,
24-0Hase (Chand] er
C't
calbindin-D(28K,9K)
(Desplan, Brehier et: al, 1983; Desplan, Thomasset et al, 1983; Perrel et
al,
1985;
Wasserman et al,
1985)
have
been studied in sorne
a1though others like1y exist (Hobden et al, The
precise
translocation
role
of calbindin- D
is eontroversiai
(see
22
in
vitamin
beIow:
Priee et al,
1980;
D-dependent
1.6.2.
detail, 1980).
calci urn
Non-Genomic),
and
1 1 1 1 1
1 1 1
1 1
there is sorne suggestion that i t may play only a subsidiary part if any in the overall response. Moreover, calbindin-D 28K i5 present in Purkinje cells of rat cerebellum despite the absence of l,25(OH)2D3 receptors in these cells
(~chneeberger
et al,
1985), and calbindin-D 9K in rat uterus
is dependent on estradiol rather than 1,25(OH)2D3 (Delorme et; al, 1983). In view of the importance played by 24-hydroxylation in the inactivation of 1,25(OH)2D} (see above: 1.2. Chemistry and Metabolism
of
Vitamin D)
induction of the 24-0Hase by this sterol in its target cells, presumably represents
a negative
feedback mechanism for l, 25(OH) 2D3 action.
Intracellular 1,25(OH)2D3-receptor concentration also is regu1ated via a receptor-mediated induction meehanism (Costa et
al,
1985,1987).
Receptor occupancy by l, 25(OH) 2D3 or other vitamin D analogues resul ts in an actinomycin-D-sens itive inerease (homologous up-regulation) feedback mediating
mechanism the
for
effeet
in 1,25 (OH) 2D3 receptor dens ity
constituting a
1,25 (OH) 2D3' correlates
eomp1imentary positive
The potency of a metaholite
with
its
relative
affinity
for
in the
l,25(OH)2D3 receptor.
1 1 1
1 1 1 1 1 1
1.6.2.
Non-Genomic.
Not aIl aspects of vitamin D action can be
8ccounted for by this genomic model. Many effeets occur too quickly to be a consequence of de nova protein synthesis,
and are not b10cked by
inhibitors of transcription and translation. Additiona1 mechanisms have been proposed, most notably the "liponomic" model developed by Fontaine and ac;sociates (1981); however, a comprehensive understanding of vitamin
D action at the cellular level is 1acking. For
a
time,
the
postu1ated
involvement
of calbindin-D
in
l,25(OH)2D3-stimulated intestinal calcium transport was held as a paradigm for the
genomic mechanism of action of this sterol.
In 1976
23
1
.1
1 1 1 1
however,
1 1 1 1
1 1 1 1 t
,
1 1 1 i
et al
demonstrated that enhanced
appearance
of
calbindin- D or
uptake
preceeded
the
po1ysomes,
and decayed faster than ca1blndin- 0
isolated,
perfused normal chick duodenum preparation,
of cale ium
cal bindin - 0 - spee if i c Employing a vascularly Nemere
f't
li1
(1984) showed that increased calcium absorption actua11y occured within on1y 8-14 min of exposure to picomo1ar concentrations of l,25(OH)203' Bik1e
J
Spencer
al
et
intestinal
(1978)
calcium
ca1bindin-0
was
further
demonstrated
transport
in
inhibited
by
chicks
that
1,25 (OH)203 - stimulatecl
persisted
actinomycin-D
when
and
syntlH'sis
of
cyclohexamide.
1,25 (OH) 203 -enhanced entry of calcium into brush-border membrane vesic1es
(BBMV) prepared from vitamin O-deficient chicks similarly was
unaffected by cye10hexamide pre-treatment (Rasmussen et al, was
proposed
dependent
that
a
1979).
It
ca1bindin-D-faci1itated
transce11ular
calcium
transport
mediated
via nuclear
genr'
activation, was preceeded by a more rapid phase of calcium uptake, which took place at the brush- border membrane (BBM). This
initial influx
WélS
rate-limiting and independent of de novo protein synthesis (Rasmussen et
al, 1982; Wasserman et al, 1982); Nemere et al (1984, 1986) called this rapid response to 1,25(OH)2D3 "transcaltachia". The precise mechanism of transea1 tachia is not clear. a
biphasic
dose
dependency
(maximal
at 650
pM;
l t displays
inhibitory at higher
doses), and occurs only when sterol is applied to the serosal surface of intestine; Putkey
et
intraluminal l, 25(OH) 2D3 has no effect (Nemere et al,
al
(1986)
demonstrated
no
BBM
de ri vati ves from radioactive precursors.
binding of
Thus,
any
198f~).
sterol
a 1 though the fi ntd
transcaltachic effect actually takes place at the BBM, its initiation by 1,25(OH)203 occurs se1ective1y at the contraluminal surface imp1ies a
multistep process,
and raises the
24
This
possibility of a surface
1 1 1
J
receptor for there
l,25(OH)2D3
is no direct
enterocyte basal membrane.
evidence for
such
a
vitamin D-binding globulin (Gc-protein)
receptor,
recent
A1though
reports
of
in tight association with the
plasma membrane in a variety of cell types (Petrini et:: al, 1983; Machii et al, 1986; McLeod et al, 1986;
Nestler et a ... , 1987)
is intriguing in
this regard. Filipin,
:1
on the
a
1ipophilic
po1yene
antibiotic
that
interacts
specifically with membrane cholesterol, mimics the effect of l,25(OH)2D3
j
on BBMV in that it enhances the transport of calcium into BBMV prepared
-1
changing membrane cholesterol content or altering Na+ - dependent glucose
from vitamin D-deficient but not vitamin D-replete chicks without
uptake (Rasmussen et al, 1979; see also Wong et al, 1975). Cis-Vaccenic
1 ] _1
acid, another exogenous 1ipophilic agent which is known ta partition to the
fluid
J ]
and
therefore promo te membrane
fluidi ty,
increases calcium uptake by BBMV from vi tamin D- defic ient chicks, not
vitamin
ac id,
1
domain,
which
D-replete chicks (Fontaine parti tions
to the
solid
et al, domain
19B1).
a1so but
Trans-Vaccenic
and reduces
membrane
fluidi ty, has no effect on calcium transport into BBMV from vi tamin Ddeficient chicks, but reverses the increased calcium uptake observed in BBHV from l, 25(OH) 2D3 - treated animaIs These
findings
suggest that
1,25 (OH) 2D3
enhanced calcium entry
across the enterocyte BBM by increasing membrane fluidi ty. In support of
1
1 ,1
this
1,25 (OH) 2D3
J 1
Matsumoto et
al (1981) demonstrated that
treatment of vitamin D-deficient chicks increased the
relative phosphatidylcho1ine (PC)
content in BBM,
with a preferential
incorporation of arachidonate over palmitate into the new1y sythesized
PC.
1
"1iponomic" mode1,
Fractional increases in unsaturated fatty acid groups in membrane
phospholipids are associated with increased membrane fluidity, and the
25
1 1
1 1 1 1
1 1 1 1 1 1 1
,
, f
1 1 1
time course with
of these changes in BBMV lipid structure
the observed changes
propose
that
in calcium transport.
enhanced
membrane
phosphatidylcholine content is a
correlated weIl
Hirata et al
f1uidity
via
common step in many
(1978)
increasE"d
surface - receptor
mechanisms. Direct measurement of membrane f1uidity by electron-spin resonnncE" by Putkey et; al between BBMV
(1982) however,
failed
to demonstrate any differencE"
prepared from vitamin D-deficient
and vi tamin
D- repletc'
chicks, and in contradiction to the findings of Rasmussen et al (1979), Bikle
et al
(1984)
found
both cis-
that
increased BBMV membrane fluidity.
and
trans-vaccenic acid
Moreover, BBMVs from both V!. tamin D-
deficient and vitamin D-rep1ete groups exhibited identical phasetransition process)
temperatures
indicative
for
calcium
of similar
uptake
fluidity
(a
states
temperature-dependent (Bik1e
198 /1).
et al,
Measurements of membrane fluidity however, are probe dependent,
and must
be interpreted with caution. Whether a1tered BBM f1uidity is the hasis for transcaltachia remains therefore uncertain. In light of the demonstration by O'Doherty (1978)
that 1,25(01l)2D3
increased the activity of ca1ciwn-sensitive phospholipase A2 • the
recent
report by Bergstrom
et al
(1984)
that
and thc'
indomethacin or
acetylsalicylic acid b10cked cis-vaccenate -enhanced calcium uptake bone,
but not that
enrichment of BBM which
is
the
eicosanoids
stimulated by prostacyc1in,
1, 25(OH) 2D3 - s timu1ated
arachidonic acid content (Matsumoto
principal
may be
precursor
involved in
for
by
prostag1andins,
et al,
198] ) ,
suggests
this action of 1,25(OH)2D3'
tha t
ThU,
however, remains to be confirmed. A similar transca1tachic effect has been reported for PTH (Nerner!-
et al, 1986). The effect was specifie for hormone applied to the serosa1
26
1 1 j
membrane, and not reproduced by the inactive peptide analogue PTH 3-34. The effective concentration of PTH in these experiments
(130 pM) was
similar to that of 1,25(OH)2D3. It is interesting that whi1e this concentration is within the physiological range for both hormones, since 99% of 1,25(OH)2D3 in plasma is bound to Gc-protein in vivo (Bouillon et al, 1981), the transca1tachic concentration of 1,25(OH)2D3 is actually 2
orders of magnitude greater than the free plasma concentration of this sterol
(1
pM).
This questions the physiological
1,25 (OH) 2D r stimulated transcal tachia,
significance of
and raises the posibili ty that
PTH is the actual mediator of this effect ln vivo. Other nongenomic effects of 1,25(OH)2D 3 have also reen described.
1
Edelman et al
1
1
(1986) reported rapid «
1 min) changes in membrane
potential (7 mV depolarization) in Necterus kidney proximal tubule in response to 100 pM 1,25(OH)2D3 applied to the lwninal surface; 250HD 3 , 24,25(OH)2D3' response.
estradiol and testosterone each produced a
50% smaller
The physiological implications of such effects are unc1ear.
Numerous nongenomic effects have been described for other steroid homones
(reviewed in Duval et al,
1983;
see also Pasqualini et al,
1986).
1.7. Parotid Gland Anatomy and Salivary Composition
t The gross anatomy of the parotid and submandibu1ar glands of the rat is depicted in Fig. 1. The submandibular glands are a discrete pair of midline structures in the neck located on either side of the trachea
1
1 1 1
at the level of the thyroid cartilage. The main excretory duct exits the rostral pole of the gland superficial to the posterior belly of the
27
1
1 ,-1
Figure 1.
Gross Anatomy of the Major Salivary Glands of the Rat
1 1 1 1 1 1 1 1 1 1
1
1 1
1 28
1 1 1
1 1 1
1 1 1 1
/, /
'/f
/ /,
l,
1
1 ,1
"
1
1
1
E (traorbltal lacrlmal gland
\ 4;
Mangos et al, 1966ab) can thus be accounted for by a diminished contact time
of the
duct.
rapidly moving saliva with
Striated cells derive their name
appearance
reflecting deep
invaginations
the resorptive surface of thf' their
from
light microscopie
vf the basal
plasma
separating tall columns of mi tochondr ia - filled cytop lasm: discernible
with
epithelia.
In
the
electron microscope,
contrast
to
the
behavior
and of
membrane· fea ture!:.
typical
of absOrpliv('
Na+
saliva,
in
rh(·
concentration of K+ decreases from about 20 meq/l at low flow rates ro levels
approaching
those
of primary saliva as
flow
increases
Th1~.
indicates that K+ is secreted rather than absorbed by the parotid duct. Ductal secretion of K+ ls more marked ln submandibular e1and (Schneyer,
1975),
while
Na+
ls handled similarly by both glands.
35
Thus,
a~.
1 1 1 1
increasing flow rate exceeds the mechanisms,
the
capacity of ductal
composition of final
saliva asymptotically approaches
that of primary saliva. lt
is implicit in this
model
gland that water enters only at the saliva,
of fluid secretion by the parotid level of formation of the primary
and is not significantly reabsorbed by the ducts.
of indirect evidence support this assumption
~
1
t 1 1 1
1 1 1
,
1 1
1 1
Several lines
Based on ultrastructural
analysis of the major epithelial constituents of the parotid gland and their relative permeabilities to a le ad ion tracer, Simpson et al (1984) concluded
that
the
inter-acinar
tight
junctions were,
in
fact,
relatively "loose" and highly permeable. Those between the lining cells of inLercalated ducts were significantly less permeable, and only those of the s triated ducts findings
truly
"tight"
and Impermeable to tracer.
These
are consistent with a pot.ential paracellular pathway for
transepi thelial fluid movements exclusively at the level of the acinus and poss ibly intercalated duct.
Moreover,
in studies using retrograde
perfu5ion of mercuric chloride to produce selective ductal damage to the s triated portions of the parotid gland system, only the hypotonici ty of the subsequently secreted saliva was t
impaired; there was no change in
he final volume (Burgen, 1967). Pr imary saliva also contains
small
nonelectrolytes, such as
glucosl' (believed to enter saliva by a combination of passive diffusion élnd sol vent drag with 1987)),
~
modification
the stimu1ated flow
and a variety of proteins,
of water
(Howorth et:
al,
the vast majority of which are
prefOll11f'd and secreted directly by acinar cells via exocytosis. Although much Ipss dramatic than its effects on fluid and electrolyte secretion, dlletal modification of the organic phase of saliva also occurs. There is
evidpncE'
to suggest that a smal1 amount of prote in is secreted by tbe
36
1 1 .1 1 1 1 1 1
intercalated and striated ducts (Lima et al, 1977; Hand, 1979), and that the striated ducts are capable of removing certain proteins from salivlI byendocytosis (Hand et al, 1987) .
1.8.2.
Salivary Calcium Secretion. As
parotid gland secretes largE'! amounts
previously mentioned,
of calcium,
although
mechanism of this process is not thoroughly understood.
the
the precise
Wallach et al
(1971) demonstrated that calcium present in zymogen granules is releasE'd in conjunction with secretory proteins during exocytosis.
Accordingly,
calciwn secretion in response to sympathetic stimulation is accompanied by a parallel decrease in cellular calcium content. evoked saliva however,
has
a
Parasympathetically
similar concentration of calcium
(5 -la
meq/l) even though very little exocytosis or loss of cellular calcium 1977,
1978).
1 1 1
translocation are utilized by the parotid gland depending on the type of
indicates that
1
The
cellular mechanism underlying the non-exocytotic
pathway is not known.
1.8.3. Neural Control of Secretion. Parotid gland secretion in thp rat is regulated by both divisions of the autonomie nervous system. ganglionic parasympathetic fibers travel from the the
parvocellular
ganglion
1 1 1 1 1
This
two different pathways fur transepithelial calcium
stimulation used.
1
1
these conditions (Schneyer
et al,
occurs under
reticular
formation
in
the
sali vary nucleus ln
brainstem
(Michell et al, 1981), which lays near
Pre-
to
the
otic
the foramen oval"
through which the mandibular division of the trigeminal nerve (V 3 ) exl thosphorylation of
phosphorylation
this protein.
substrate
to
The
protein
secretion, as weIl as its identity as the critical convergence point of
f
the PK-C and PK-A oathways is therefore suspect.
t
J
1
56
1 1
1
1 1 i 1 1 1 1
1.10.3. Calcium and t:he cAHP Pathway. Forskolin stirnulates cMtPrnediated protein
secretion by
a
direct
action
on adeny1ate
cyc1as€',
bypassing the p-adrenoceptor (Watson et al, 1983; Mednieks et al, 1986) However, the secretory response of rat parotid gland to lO",M forskol i is approximately half that
of ll-'M isoproterenol,
doses
a similar
both
agents
(Dreux et al, invol ved
in
produce
1986).
the
This
incrE'ase
even in
though
st tlWSt'
intracellullll
cAHl'
indicates that 8n additional messenger
,B - adrenoceptor -mediated
response.
Several
11
is of
lines
evidence suggest that calcium may serve as this messenger. Unlike prote in secretion ~-adrenoceptor-mediated
normally in
in
response
exocytosis by
the
to cholinergie agonists. parotid
gland
proceed~
the absence of extracellular calcium for prolonged pE'riods
of time (Butcher et: al, 1980; Argent et: al, 1985; Dreux et al,
1986)
Calcium appears, nevertheless, to play sorne role since undE'r condit ionnts with methylprednic:;olone (1979);
J
pmol/g
or
(l mg/rat)
500 as
pmol/g),
or
5
in Tenenhouse
daily et al
steroids were dissolved in 0.1 ml ethanol (250HD 3 , 24,25(OH)2D3
or 1,25 (OH) 2D3)
or
propylene
glycol
(D 3
or methylprednisolone),
and
adll1ini.stered by ip injection.
2.3. Collection of Saliva
Rats were anesthetized with sodium pentobarbital ln
prE'llIninary
hvpothermia;
J J
experiments, salivary
charac terlstically slow
flow
rates
in
rats
often
these
susequent experiments,
developed
animaIs
core
ip).
were
temperatures
werp contlnuously monitored with a small rectal probe, and maintained at 37 oC ± 0.5
oc
using a homeothermic blanket system (Harvard Apparatus
Co , South Natick, MA)
1 1
In
anesthetized
(50 mg/kg,
In sorne animaIs, heart rate and blood pressure
64
1
1 1
were
continuously monitored through
polyethylene catheter (PE 50, saline
(1000
transducer
units/l),
and
a
Grass
the
left
Clay Adams,
and attached to (Model
7)
femoral
NJ)
artery
using
a
filled with heparinizpd
a P23Db Statham prpssurp
polygraph.
Tracheostomies wer{'
performed on all animaIs to prevent aspiration of saliva, and the le ft
1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1
jugular vein
was catheterized wi th
PE 50
polyethylene
tubing
for
iv
injections. The right parotid duct was behind
the corner of the
facial nerve (Fig. 2)
1
approached through
a
small
incision
identified at the bifurcation 01 the
mou th ,
and isolated by blunt disection.
A ') cm cannula
(PE 10 polyethylene tubing) prepared with a bevelled tip and containing a thin wire for support was inserted a short distance flute-hole incision made in the side of the duct.
through a
sma11
The wire was remnvcd,
and saliva collected in tared microcentrifuge tubes at 10, 20 and 30 min post- stimulation time
intervals.
In sorne experiments 1
saliva was also
collected from the main excretory duct of the ipsilateral submandibular gland cannulated midway Along its course beneath the anterior be lly of the digastric muscle (Fig. 1). Salivation was usually stimulated by iv administration of a sin81(' dose
of
pilocarpine
dissol ved in
0.1
ml
isotonic
saI ine;
a
maxima 1
secretory dose (4 mg/kg body wt) was most often employed. ln one set of experiments
however,
parotid
gland
salivation
was
evoked
by
direct
stimulation of the postganglionic parasympathetic nerve to the gland. In these experiments, the ipsilateral auriculotemporal nerve was exposed by blunt disection as it emerged from behind the neck of the mandible pulses
of electricity were delivered at a
frequency of 20 Hz
4 mV
(5 mser
duration) to the unsectioned nerve through a bipolar platinum electrorlp using a
sn
Both the nerve and electrode wen·
5 Grass stimulator.
65
submerged in paraffin as an insulator, and the surrounding soft tissues further insulated with Parafilm AlI saliva samples were weighed immediately, and stored at -20
oc.
Volumes were caleulated from density relationships determined in preliminary studies (parotid and submandibular saliva both had a density of 1 mg/fll, rats younger
independent of flow rate) . than 49
morpho logically
days
(Winick et
of age, al,
Saliva was not colleeted from as
1965;
the parotid gland is Schneyer
et al,
1969)
not or
fune tionally (Schneyer et al, 1968) mature before this time in rats. At
the
end
of
eaeh
experiment,
the
rats
were
killed
by
exsanguination through the abdominal aorta, and the right parotid gland and submandibular glands excised and carefully trimmed of fat and lymph nodes in isotonie saline.
Gland wet weights were obtained following
brief centrifugation (10 000 x g for 10 sec) to remcve 100se1y adherent saline
The rate of saliva production was expressed as fll/min-g gland
tissue. Average flow rates and total outputs were calculated of 60 min and 90 min collection periods respectively.
in terms Blood for
measurement of serum constituents was obtained from the abdominal aorta.
2.4. Measurement of Serum and Saliva Constituents
Amylase activity was measured with the Phadebas Amylase Kit (Phurmacia, Uppsala,
Sweden) by the method of Ceska et al (1969).
Calcium measurements were made using the Calcium Rapid Stat Kit (Lancer, a division of Sherwood Medical,
St Louis,
MO),
as \vell as by atomic
absorption spectroscopy using a graphite furnace;
the two rnethods
yielded identical results. Inorganic phosphorus in serum and saliva was
66
J
~-----------------------------------------
J 1
1
1 1 j
1 1 1 1
1 1 1 1
, ,
determined colorimetrically using the American Monitor Phosphorus reagent system (Fisher, Ottawa, Ontario). PTH measurements were made using a mid-molecule radioimmunonssny kit (Immuno Nuc1ear Corp., Stillwater, MN). Serum 250H0 3 was measured by radioimmunoassay
(Immuno
1 1
The
limi t
of detection
for
250H0 3 was 0.5 nmol/l serum. Serum concentration of l, 25(OH) 2D3 was measured with a calf thymus receptor
competi tive- binding
assay:
receptors were
from calf thymus as described by Reinhardt et al
partially purified
(1982), and incubllted
at 5 oC for 15 h with 1 nM [3H]l,25(OH)203 ± either l,25(OH)203 purified from
se rum,
or
various known
concentrations
of non- radioac t ive
l, 25(OH) 2D3; free and receptor-bound sterol were separated with dextrancoated charcoa1
Sterol Binding).
(see below: 2.11.
Sucrose Denslty GradIent Analysis of
In initial experiments, 1,25 (OH) 2D3 was extracted frolll
serum with chloroform:methanol (2:1), dried under N2 , redissolved in 0.5 ml
n-hexane: isopropanol
(90: 10) ,
and
separated
from other vi tamin D
metabolites by HPLC on a Zorbax Sil co1umn (Dupont, developed
in n-hexane: isopropanol
(90: 10).
Boston,
MA)
Later experiments employed
ether to extract serum lipids, and included an initial purification of l,25(OH)2D3 on silicic acid columns in ether:n-hexane (80:20) prior to HPLC separation
in n-hexane:isopropanol:methanol
(94:5:1)
on a
Zorbnx
Sil column. Similar results were obtained with both methods. The limJt of detection for
1,25 (OH) 2D3
in 10 ml samples of
poo] ed
serum
was ?
pmol/l serum. Serum 24,25(OH)2D3 was measured by a modification of the method of Fraser et al (1986)'
serum lipids were extracted in methanol:methyh'IW
chloride as described,
1
Nuc1ear Corp.).
24,25(OH)2D3 was
then separated from otlwr
vitamin D metabolites including 250HD 3 -26,23-lactone by sequential HPLC
67
1 1 1 J 1 ,1
J 1 1 J ]
(positive-phase HPLC on a Zorbax-CN cohunn (Dupont, Boston, MA) e1uted in n - hexane : isopropanol: me thanol HPLC
(94: 5: 2) ,
on an A1tex Ultrasphere ODS
methanol:water competitjve saline) as
(4:1»;
24,25(OH)2D3
protein-binding a
HPLC-purified
source
column
assay
]
by
reverse -phase
San Ramon,
CA)
dilute
[3H]250HD 3 and 24,25(OH)2D3
in
purified was measured by rat
serum
(1/10 000
of vitamin D-binding g10bulin (Gc-protein), as
competing
ligands.
and The
limit of detection for 24,25(OH)2D3 in 6 ml samples of pooled serum was 0.03 nmol/1 serum.
2.5. Renal 250HD 3 -24-Hydroxylase Assay
Kidneys of normal and G2 Ca+/D- rats were excised, and homogenized in 5 volumes
of a buffer containing HEPES
(20 mM,
pH
7.4 at
21 OC),
sucrose (190 mM), MgS0 4 (2 mM), EDTA (1 mM) and malic acid (20 mM) using a Potter-Elvehjem homogenizer with
a Teflon pest1e.
homogenate was incubated wi th 500 pmo1 [3 H] 250HD 3 oC wi th continuous agitation for 30 min.
,1
(Beckman,
so
using
followed
l
ml of kidney
(150 pCijmmol) at 25
Sterols were then extracted in
chloroform:methano1 (2: 1) and saturated KCl, dried with N2 and separated by HPLC in n-hexane:isopropanol:methane (95:4:1) on a Zorbax Sil column. Renal 24-0Hase activity was expressed as pmo1 24,25(OH)2D3 produced per
1
mg protein in 30 min.
1 1
1
t 1
2.6. Amylase Secretion In Vitro
Rats were killed at 12-20 weeks of age by exsanguination through
68
1 1 1
1 1 J
1 1
1 1
1 1 1 1
,
, 1 1 1
the abdominal aorta under light ether anesthesia. Parotid glands from 35
rats at a
minced
in a
time were pooled,
trimmed of fat
and lymph nodes,
Krebs-Ringer bicarbonate buffer (KRB)
hydroxybutyrate
(Babad,
Ben-Zvi,
glucose as energy sources,
Bdolah & Schramm,
and l
mM calcium.
preincubation in 40 ml KRB at 37 oxygenation (95% °2/5% CO 2 ),
containing 1967)
and
5nlf>t
f3-
and 0.3%
Following a
15 min
oC with agitation and continuous
100-250 mg samples of parotid tissue were
incubated in 25 ml erlenmeyer flasks in ei ther 4 ml KRB containing 1 mM calcium, or 8 ml calcium-free (no
added calciwn) KRB (CF-KRB).
Isoproterenol or carbachol (10 pM) dissolved in 50 pl buffer were added directly to the suspen'3ion medium after a 15 - 30 period; elici t
the
dose
used was
determined in
preliminary
a maximal release of amylase from parotid
of animaIs.
In sorne
experiments,
min stabi lization
propranolol
(1
experiments
ta
tissue in all groups J.'M)
was added 3 mi Il
after or 20 min prior to stimulétion with isoproterenol. Aliquots interva1s,
of medium (1%
and assayed for
t'lta1
';olum,~)
calcium
(only
were
sampled at 15-30 min
ll.cubations
in CF-KRB)
and
amylase content as pre'lious1y described (see above: 2.4. Me8surement of
Serum
and Saliva
expressed as
Constituents).
Amylase
and
calcium
secretion werc
percentages of the total glandular content calculated by
adding the total amount secreted to the residua1
glandu~
ar amylase in
homogenates of the f1ask contents at the end of each experiment.
Eacb
~
experiment was repeated at least 3 times,
al though sample times varied
slightly
2.7. Carbacho1-Stimulated K+ Efflux In Vitro
Parotid glands were excised,
69
and minced in
KRB containing 1 ml1
--~
1 1 1 1
1 1
t J 1 1 1 1 1
1 1
,
calcium as before.
-------------------------------
Approximately 30 mg of parotid tissue was placed in
each of up to 10 superfusion chambers of the apparatus i1lustrated in Fig.
6,
and superfused
(verified using a
(0.5 ml/min) with buffer maintained
be highly temperature sensitive). (3 Jl.Ci/ml)
1
Parotid fragments
shown to
were loaded wi th
or 42 K (5 /.ICi/ml) in KRB for 40-60 min (Fig. 6a);
preliminary experiments demonstrated saturation of tissue from normal and vitamin D-deprived rats within this
time.
Tissue was subsequent 1 y
superfused with tracer-free KRB ± calcium, and 1.0 ml samples collected at 2 min intervals for 50 min, and counted by 1iquid scintillation (Fig. 6b) . In sorne experiments, sample and ana1ysed for agents
were
added
100
amylase
directly
to
J.ll aliquots were wi thdrawn content. the
Secretagogues ltnd other test
At the end of each experiment,
from
homogenized in 2.0 ml 0.1
radioac tivi ty.
Res idual
amylase
following homogenization in water. were tubes.
determined
after
fo11owing
formula:
k
content of
=
tissue was removE'd
N HCl,
and counted for
the tissue was
measured
Wet weights of parotid gland tissue
brief centrifugation
Coefficients of efflux for
an
(86 Rb or 42 K efflux stabilized
within 12-14 min). the chambers,
from ea"h
superfusion buffers following
initial 20-24 min equilibration period
in
tared
microcentri fugE'
86Rb and 42 K were calcu1ated hy tlw
61n%/ ôt,
where
lün% was the change in thf'
natural logarithm of the percent radioactivity remaining in the ti ssue at
the beginning
of each
interva1,
interval in min.
1 1
oC
miniature temperature-sensitive probe placed wit lin
the chambers; monovalent cation-nonspecific channels have been
86 Rb
at 37
70
and
Ât was the
duration of the-
1 1
1 1 Parotid Gland Superfusion Apparatus
Figure 6
Parotid gland slices were housed in 300 Jll chambers
canstructed from the
tips
superfusian
of plastic 1.0 ml syringes placed
back- to - back and held together by a small s leeve of rubber tubing. The ends were
fitted
with
hypodermic
needles
connected to
tubing
Oxygenated KRB containing radioactive tracer was
ml/min)
through
a heated water bath
temperatures at 37°C.
to maintain
10 chambers could be
polyethylene purnped
(0.5
superfusion chamber
superfused in parallel. A.
During t Lssue loading wi th radio isotope , buffer was cycled through the reservair far 40-60 min. B. Afterwards, tissue was superfused with fresh KRB 1 and l ml samples collected with a fraction collectar.
71
J 1 1
1 1 1 1 1 1 1 1
1 1 1
,
1
1 1
A.
1
tissue
1
chaJnber
j
lit
M
1 1
peristal tic pump
1 1 1 1
B.
1 tisS'Tle
1
ch aJrtl:ler
f"I. ..
J
1 1
, 1
agonist
.
'.
. '.:.. ': ~f"VA
cpm
••••
~'t.AJ
a
peristaltic pump
. ~
1 1
2.8. Preparation of Cytosol from Whole Tissues
j
w~re
Normal rats (200-300 g)
exsanguinated under light ether
anesthesia by incision of thE' abdominal aorta. The duodenum was removed,
4 J
1 J
1 1 J
flushed with ice-ccld calcium and magnesium-free KRB (pH 7.4 at 20 OC) (CMF-KRB),
] '.1
i ts
length.
Mucosal cells were scraped
free with the edge of a glass slide, washed with Ct1F-KRB, and collected by
centrifugation
(50 x
g at 4
oC x
5 min).
Other
tissues
(parotid
gland, submandibular gland, pancreas. liver and quadraceps muscle) were simply excised, trimmed of fat and lymph nodes, and All
subsequent steps were
carried out
mince~
at 0-4
oC.
in CMF-KRB. Tissues
were
homogenizeè in 2-3 volumes of a buffer (KTEDMo) containing KCl (300 mM), Tris-HGl (10 mM;
pH 7.4 at 20 OC). EDTA (1 mM),
dithiothreito1 (SrnM).
sodium molybdate (10 mM) and phenylmethylsulfonylfluoride (0.1 mM) using a Brinkman Polytron blender or wi th
-1
and cuL open along
a Teflon pestle.
a
Potter-Elvehjem homogenizer equipped
Preparations
were
periodically cooled on
ice
during homogenization in order to avoid receptor loss due to excessive hea t ing,
Supernatant
cytosolic
fractions
(8 -10
mg
proteinjml)
were
obt:.lÎned by centrifugation (100 000 x g at 4 oC x 60 min). Prote in was measured by the method of Bradford (1976) using crystalline bovine serum ôlbumin as a standard.
_1
l l 1 J
1
2.9.
Preparation of Nuclear Extracts from Parotid Gland Tissue
Minced parotid gland tissue from 15 rats was homogenized in 5 ml TEDMo buffer (KTEDMo containing no KCl). Nuclear pellets were obtained
72
1
by centrifugation (800 x g at 4 oC x 15 min), and incubated on iee for 5
1 1 1 1
min
1
2.10. Preparation of Cytosol from Iso1ated Acinar Ce11s
,
TEDMo
eontaining 0.05%
washed 3 times wi th 10 ml
TEDMo
extracted wi th 2.5 ml KTEDMo for period,
Tri ton X-IDa.
Preparations W(>l"\.'
containing 250 mM sucrose, 30 min on ice.
At
and then
the end
of
this
chromatin and debris were removed by sequentia1 centrifugation
(800 x g for 15 min, then 100 000 x g for 60 min) at 4 oC.
1
1 1
in 5 ml
Parotid gland acinar cells were isolated by a modification of the method of Mangos et al (1975). 30 parotid glanrls were pooled and minced in CMF-KRB at room temperature. AlI glassware was routinely prior to use.
Minced tissue was
~i1iconiz(>d
ineubated in 60 ml CMF-KRB containinr,
eollagenase (130 ID/ml) and hya1uronidase (77 lU/ml)
for 60 min st 37
oC, with eontinuous shaking in an atmosphere of 02+C02 (95%:5%).
At the
end of this period, ee1ls were meehanieally dispersed by repeated
1 1
1 1 1
,
aspiration with a 10 ml pipette having a bore of 2mm.
Cells were
collected at room tempe rature by centrifugation (50 x g for 5 min), and incubated in CMF-KRB eontaining trypsin (50 lU/ml) at 37 oC for 10 min Following a second dispersion, the suspension was filtered through a J1.m
nylon mesh,
and washed twice with CMF-KRB.
method typically displayed
Cells
4~
isolated by thic,
greater th an 99% viability as indicated by
exclusion of 0.4% trypan blue dye. Pancreas
aeinar
cells
eollagenase/hyaluronidase-digested
were
prepared
from
tissue as deseribed by Chauve] ot et
al (1979).
1
1 1
Isolated parotid or pancreatic acinar cells were
73
homogenized
011
Isolated parotid or pancreatic acinar cells were homogenized on ice in 2-3 volumes of KTEDMo using a Potter-Elvehjem homogenizer with a Teflon
pestle.
inspection of
Cell
lysis
was
the homogenates.
confirrned by
routine
microscopie
Cytosol was subsequent1y prepared as
described for the whole tissue homogenates.
2.11. Sucrase Density Gradient Analysis of Sterol Binding
200 ,Jl aliquots of cytosol (2-10 mg protein/ml) or nuclear extract were ineubated
for 4-20 h at 4 oC
in Eppendorf microcentrifuge tubes
containing the
appropriate st( roIs
dissolved in
10 III
ethanol
(ip a
number of experiments tubes were dried of ethanol under a stream of N2 before
addition of eytosol;
this
did not
Unbound sterols were removed at the
alter
final results).
end of this period by addition of
1/10 volume of dextran-coated eharcoal solution A and 0.05% dextran T-70 in KTEDMo).
the
(0.5~
acid-washed Norit-
After 15 min at 4 oC, charcoal was
removed by high - speed centrif'.lgation. Sarnples were 1ayered anto chilled 5-20% continuous suc rose density gradients prepared in KTEDMo using a
Beckman gradient former (Beckman
Instruments, Fullerton, CA). and centrifuged at 300 000 0-4
oC.
par.111el
Chymotrypsinogen (2.5 gradients
as
S)
and ovalbumin
sedimentation
markers.
g for 15 h at
X
(3.7 S)
150
pl
were
run on
fractions
were
collected from the bottom of each tube, and assayed for radioactivity by 1iquie! scintillation.
In one set of experiment.C
:::!1l
4
1-
l\\,.
u
2
1
1 1
c. o.
10
CL.
1
10 A
.\.....f,l-I
l-
0
c
L.
c.
1
-
N 1
-0
:::!1l
1
1
4
CL.
u
2
2
1\ 1 -
,
l'
\.~r
5
6
1
"'~
-
\
,
10 15
20 25
r
""""""",,,,,,{
l'
l'
Fraction No.
10
N 1
...
0
.... )(
4
\ 'J.
5
0 10
1
"\")",,,.:..~ ..~)
o
0
~
6
>C
~ Q,
O.
1
• •
8
4
........... _.. _..J ......_.... 0
c. o.
0
)(
~. .\ 1
•
...
>-o_ ...
15 20
~
Q,
0
2 ')
..!
25
1 1 1 1 1 1 1
Whole Parotid Gland Cytosol
A, 1.0 nM [3H11,25(OH)2D3' B, 1.5 nM [3H124,25(OH)2D3' C, 1 0 nM r3H]1,25(OH)2D3 + 50 nM 24,25(OH)2D3; 0,
+ 500 nM
[3 I1 )l,25(OH)2 D 3
1,25(OH)2D3'
r 3I1 )24,25(OH)2 D3
+
3 [ H)24,25(OH)2D3
500 nM 24,25(OH)2D3'
t
500
nM
1,25(OH)2D3;
e,
l.0 nM
D,.,
l.5
nM
0,
l.5
nM
1 1
1 1
1 1
1 1 1
1 87
1
1
1 J
1 J
:4-
f
,...
§
1 1 1
1 1
J4CO c;lm
1 ~O
J:: 5
16~
:., s
1
........
1
: '.
1
)(~::..., 1
::::
1
1.,;
e-
Il.
! 1
/- il 1 1
ï\--~/: a
a
·
\ le
12
24
1
,
1 J
1 1
o
i
1
4400 cpfft
1
20-
le
12 Frcc~
on
.4
12
FractIon
24-,
o
6
FractIon
J 2 S
1 1 1
-.
cc
1 1
&000 cp""
20-
rI\
.... 1
b
2.
~
eoo 400
200
0
0-10
, 1-20
21-~0
41-60
Minutes
61-90
Figure 25
Calcium Concentration in G2 Ca-/D- Parotid Saliva Secreted ln Response
•
D , G2
, normal rats (n=5);
D,
Pilocarpine
G2 Cs- /D- rats (n-4);
Ca-/D- rats treated with D3
x 2, n=4).
t~
(100 lU, ip week1y
1 1 1 1 1 1 1 1
1 1
1 1 1 1 1 1.
107
1 1
1
1 j
J ,1
1
,
1
1 1 J
10
-
8
. .J
""
0' W
E
-E
6
.-u:;,
-
4
ca
(J
1
2
1
o
,
1
,
, 1
1
o - 10
10 - 20
20 - 40
Minutes
40 - 60
60 - 90
Figure 26
Calcium Concentration in G2 Ca+jD- Parotid Saliva Secreted in Response to Pilocarpine
•
, normal rats (n-S);
D , G2
Ca+/D- rats (11-5).
1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 Il 108
1 1
1
1 J ] J
j
J
7
-":>
'5
J J
cr
e ~
QJ
E ......... E ~
'ü
C
u
.:s
f J o~--
J J
l J
f
1
1
0-10
11-20
21-40
~1-60
Minutes
61-90
- - - - - - - - - -
1 1
1
1 1
Ca-/D- diet.
normocalcemic, obtained
1
1 1
1 1 1 1 1
1
,
1 1
G2 Ca+/D-
rats was similar at a11 time poj nts to
from normal rats
(Fig
26).
This 40% decrease in
G2
1
tlH' th"l
Ca - /D-
parotid saliva calcium concentration combined with the 60% reduction in salivary flow among this group resulted in a 90 min total calcium out pu! by G2 Ca-/D- rats
± 0.6 meq/kg,
(4 1
n-j) which was
only
l/l~
that of
normal rats (17.2 ± 1.4 meq/kg, n-5) or D3 -treated G2 Ca-/D- rats (16 )
± 1.9 meq/k!;,
1
1
The concentration of calcium in parotid saliva from
n~4).
3.3.6. Effects of Different Vitamln D Hetabolites on Parotld Secretion
in Vitamin D-Deprived Rats.
Figures
27
and 28
depict
t1H'
parotid salivary response to pilocarpine observed in the G2 vitamin Ddeprived
rats
at
various
times
metabolites. As can be seen, }.ll/min-g, rats
11=4;
exhibited
Fig. a
27)
after
treatment
without treatment
and Ca-/D-
with
D)
Ca+/D-
or
(18
i!.,
±
36
(18 ± 2.1 JlI/min-g, 11-5; Fig
markeùly decreased
capacity
for
saliva
compared to normal rats (40 ± 1.8 JlI/min-g, 11-6; P Illodest re lease of amylase than did isoproterenol «40 % wi thin 2 h (Fi~
32»
Onlv basal rates were observed when carbachol was used in
112
1 1
1 1
1 Figure 29
Cumulative Basal and Isoproterenol-Stimulated Amylase Secretion from Parotid
Normal
and
Gl Ca- /D-
1
r
Gland Slices
1. A. ca 2 +-cùntaining KRB . • , normal; 0, Gl C!l-/D-. B
ca 2 +-free KRB . • , normal; 0, Gl Ca-/D-.
Values are means
±
S. E. from 3 replicate experiments.
1 1
1 1
1 1
, 1
113
1 1
,
1 1 1 j 1
,
1
100
1
-::::::- 80
1
1 1
, :1
l
,
1
,
1 1
.....0 ..... 0
1
100l
A.
J
~ eo~
c
---0
60-
~ Cl)
40-
,.
QJ 40-
en
c
0
>-
E
-
>-
E
...
-ex:
..- -,...
-..r- -:.--=-8
~ ~::~~::::::~:::::::.~ O~
E
~
20
20
o
i
,
1
30
60
90
Minutes
i
120
o~----------------~--~ 120 30 60 90
Minutes
1 1 1 1
1 Figure 32
Basal and Charbachol-Stimulated Amylase Secretion by Normal,
Gl Ca-/D-
and
G2 Ca-/D-
Rat Parotid Gland Slices
•
, normal, 0,
Ca-/D-.
Valups are me ans ± S.E
1
from 3 replicate experiments.
1 1 1 1
1 1
1
117
1 1 1 1 1 1
1 1 1 1
1 1 1
G2
'001 1
1 1 1 1 1 f 1 J
, 1 1 1
080 ...
o
1
....
.s ~
.........
1 1
60-,
,1 1
1
Q)
CIl
o
>,
40"':
, 1
i
E 20'"
-
BO
J!
~ 0
8
>-
20
E
20
.q;
1 :50
f 1 1
1 1 1 1
60
Minutes
90
120
:50
60
Minu(es
90
120
1 1 1
1 1 Carbachol-Stimulated 86Rb Efflux from Normal Rat
Figure 34
Parotid Gland Slices
Ha ln
Graph:
for cillculation)
86 Rb e fflux (k,
± S,E,
means
see methods
in response to 30 JJM from
10 rep1icate
imE'nt 5 lnst't
•
1
above the basal rate
Values are
ca l'bacha l P:-'1)(' r
Increase in
Respose/concentration dependency of initial
) and sustained
rarbachol,
(
0
)
phases of 86 Rb efflux to
determined at 4 min and 20 min respectively, and
f":Pl'('ss('d as mean percentages of the maximal response 1('1'] iC,ltp
1
expcriments
in two
1 1 1 1 1 1
1 1 1 119
1 1 1
-----
1
1 1 1 1
14-
f
12
1 J
...-...
1 1
1 1 1 1
1 1 1 1
-;- 100
C
0
""'eR
'"-'
--
•
phan 1
a:: 40 1
ID
CI)
6
20
0
..0 tO
1
.0
Cr::: 1
phase Il
~ so
";
8
•
fi:
ISO
)C
:::l Q)
,,-
E
°E 10 X
1
120 ]
-J
-1 0 1 2 corbochol ()Jt.A)
-2
Log
4
J
CO
:J o------.,. . ./ 1
4
~-1
--'---r- - ' - - 1"'----'-1 1 8
12
16
Minutes
20
.3 2
1 1 1
Atropine (0.1 mM)
abruptly terminated either phase of carbachol-
stimulated 86 Rb efflux (Fig. 35a). Addition of EDTA (Fig.
35b) or
removal of calcium
(Fig
36) from the superfusion buffer prevented the
sus ta i ned phase of
86 Rb
e fflux,
but only minimally
diminished the
initial transient phase. Readdition of calcium restored sustained 86 Rb efflux (Fig. 36a) in a concentration-dependent fashion (Fig. 38; maximal ~,uslained
however,
responses were achieved with 1. 0 mM calcium); this effect was, dependent on continued receptor occupancy as demonstrated by
the lack of response
(Fig
.1
36b) .
Superfusion with isoproterenol evoked a copious secretion of amyl ase
J
if calcium replacement was preceeded by atropine
from
comparatively
parotid modest
gland
response
slices, (Fig 37).
while
carbachol
In agreement
oblained using the whole animal preparation (see Sl'cret 1011
J
above:
produced
with
.1
the data
Pa.rotid Gla.nd
In VIVO) and tissue-suspension incubations (see above: Protein
Secret 10n tram Parotid Gland In Vitro), amylase secretion in to
a
response
either agonist was identical for normal and vitamin D-deprived rats . The resulrs of these preliminary experiments were consistent with
rI Ild 1 ngs
reported by oth.:!rs using identical or analogous
(l'ogroioli
et
:1
methods
al, 1982b; Putney, 1986).
3 5.2.
K+ Etflux trom Parotid G1a.nd in Vitamin D Deficiency. As
61lOwn in Fig
38, parotid glands from normal and vitamin D-deprived rats
(Ca-/D-) displayed identical 86Rb-eff1ux characteristics in response to
]
carbacho1. Moreover, the concentration dependency of the sustained phase of
efflm. to calcium was the same for both groups.
Furosemide
(0.4 pM
.1dded 8 min prio!' to stimulation) did not affect the normal 86 Rb efflux
J 1 1
Carb3cho1-stimulated
120
42 K efflux was
qualitatively and
1 1
quantitatively identica1 to that observed with 86Rb , and again, thprp was no difference between the responses of normal and vitamin rats (Fig. 39).
1 1
1 1 1 1 1 1 1
1 1 1
1
1 t 1 1
121
D-depriv~ct
1
1 1 1 1
1
1 Figure 35
Effects of Atropine and EDTA on CarbacholStimulated
86Rb Efflux from Normal Rat Parotid
Gland Slices
Increase in 86 Rb efflux above the basal rate in response to supC'rfusio'l
with
30 J.LM
carbachol
in
Ca 2 +-containing KRB.
Atropine (0.1 mM) or EDTA (1 mM) were added at the indicated post-stimulation times.
1
1 1
1 1 1 1 1 1
1 122
1 1
1
1 1 1
1 1 1 1 1
1 1 1 1
1 1 1 1 1 1
12
12
-10
-10
,5
c:
'Ë
E
"- a
~ )(
1
:=
...::: ~
6
~
~
"-
.!.8 )(
:::l
;:
Q)
6
EDTA
l
.a a:
J:l
Il:: 1
atropine
- 60
.2 60 >-
1 1
, f
, l , 1
1
.
1 1 -
120
E
E
E
lllg
Sustained 86Rb efflux in nOLmal ( . , n=3) and 0,
n-3) parotid glands measured 20 min post-
with 30 {lM carbachol in KRB containing concentrations of Ca 2+.
1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1
1 125
1 1
1 1 i 1 1 1 1 1 1 1 t
, 1
1
1
1
1
1
1 1 J 1 L
1
1 1 1 1
1 Figure 39
Effect of Vitamin D Deprivation on CarbacholStimulated 42K+ Efflux from Rat Parotid Gland
Incf('a'sp in 1I~[))
[12
K+ efflux above the basal rate in normal ( • •
ancl G2 Ca-/D- (0, n-5) rat parotid glands in response
t () ~upL'lofu'il011 wlth 30 0 (
)
d
tM carbachol
1 1 1
in KRB containing 1 mM
1 1
1
1
1
• 1
1 1 126
1 1
1
1 1 1
1 1 1
1 1
10
C8 E
""'-
~ '-'"
x 6 :::l
...... ......
Q)
1
1
1 4l
\
i.\
iJ
\~f-t -t-f hormone i..,
1,25(01l)2D3
VIa
receptors which modulsion
of
of vitdllllli
TIl('
C
1 il S '. 1 (
and Christakos et al
this organ
Because
(1981)
supported a
1
intréH'(·lllJ!;,I"
the cell
D{'ln()n~t rd! jf/Il
role
this point was so critical, l
128
il
specifie
of such recepton for 1,25 (OH) 2D) in parotid gland cytoso1 by Aoki (1979)
/)
el
.Ji
for vitarnill /)
III
continuee!
thl",('
1
1
1 1 1 1 1 J J 1 J
lnvestigations.
l repeated the experiments, and extended them to include
the acinar cell of
the parotid gland,
and other exocrine glands
including submandibular gland, lacrimal gland and pancreas. In
preliminary
the
experiments,
sucrose
dens i ty
gradient
fractionation technique employed was verified with cytosol prepared from .III
cc:,tablished 1,25(OH)2D3 receptor-containing tissue (duodenum) and two
tissues known ',keletal
to lack receptors for l,25(OH)2D3
muscle)
As
shown in Fig.
typical two component afflnity, kllown
contain
Introduction:
higher-capacity
specifie,
high-
[3Hj1,25(OH)2D3 binding in the 3.2 S fraction,
the
1.6.
duodenal cytosol demonstrated a
[3Hjl,25(OH)2D3-binding profile:
low-capacity
to
8,
(liver and mature
mammalian
receptor
for
Cellular f1echanism of Act:ion) ,
binding to a
6 S component
that
l,25(OH)2D3
(see
and lower-affinity, preferentially
bound
[ 311 J 250HD 3 , and was present in all three preparations. Sterol binding in
the
S cytosolic fraction had been demonstrated in a variety of
6
tj~~lIes,
:.lne!
was believed
to reflect
contamination
of
cytosolic
preparations by the plasma transport globulin for vitamin D, Gc-protein (Kream et al, 1979; Van Baelen et al, 1980; Cooke et: al, 1986). EXElmination of rat parotid gland cytosol using the same methods
J f
1
demonstrated the presence of a similar 3 2 S receptor for 1,25(OH)2D3 in this
tissuC'
(Figs.
9-10).
l'.lrot id rC'cpptor dlsplayed an affinity
copurified
with
the
hOl11ogenizatiol1 buffer
1
1
pl
(,~C'l\t
nuclear
(Fig
11).
elsewhere,
the
for nuclear chromatin,
and
fraction The
receptors
in
low
6 S binding
ionic
strength
component was
also
in whole parotid gland cytosol, and like Gc -protein had a lower
dfflnitv for 1,25(OH)2D3 than 250HD 3 or 24,25(OH)2D3' and did not bind t()
l1uclear
chromatin.
1 . 2S (Of!) 2D 3 recC'ptor
1 1
Like 1,25(OH)2D3
Subsequent
experiments
in cytoso1 prepared from
129
demonstrated
isolated parotid
the gland
1 1
1
acinar cells
(Fig.
1 1 1
1 1 1 1 J 1 1 1 1
,
r 1
these washed cell preparations. plasma
contamination was minimal, and cytosol was
relatively free of tht' 6 S
component.
the
Further
characterization of
dernonstrated binding specificity for (approximate1y
1
12). In
0.1
nM)
similar
to
acinar celi receptor
l,25(OH)2D3
(Fig.
that reported
14),
for the
a Kd
llnd
1,25(OI!)2DJ
receptor in classical target tissues for vi tamin D. These experiments confirmed that rat
parotid gland
aeinar
cdh
contained a l,25(OH)2D3-binding protein that was identical by c1nssiclll criteria to the 1.25(OH)2D3 receptor identified in intestine (Feldnwli et al,
(Chen et al,
1979), bone
1979)
and kidney (Colston et al.
tYr(·~.
This did not. however. rule out the possibility that other c{'ll the parotid gland a1so exists
to
parotid
date
no
gland,
sui table
contained receptors
established method
the
to address
techniques
for
1.25(01l)2D3'
iso1ating duct
emp10yed in
this question;
for
these
19HO).
M.
t1H'If'
cplls
experiments Rn'
autoradiography
(Narbaitz
III
C't
f
l'Olll
Ilot :J 1 •
1981; Stumpf et: al, 1987) would offer a more plausible approach 1,25(OH)2D3 sensitivity did not appear to be a genera1 propC'l"ty of exocrine
glands.
as
neithcr
1acrima1 gland,
submandibu1ar gland
not
acinar ce11s of the pancreas contained receptors for
1,25(OIl)ï[)~
Despite the presence of these receptors in parotid gland,
thf'
arr,dl! Wil'.
in a number of ways un1ike other target tissues for l,25(OH)2D3' not contain 24-0Hase (A
Tenenhouse &. M. Warncr,
in most l, 25(OH) 2Drdependent tissues 1978; Howard et:
al,
(Tanaka et al,
1981; Gamblin et: al,
1,25(OH)2D3
in
concentrations
enzyme more
than
100 - fo1d
in
which kidney
1974,
1985; Reichel
24-0Hase activity was not induced by
Horeover,
reported
unpublished) ,
increased
It did pn'sPllt
KUllln)"
eï al,
(lt
lCJH(J)
pre-treatrnf'lIl wltli
the
activity of
Although Goodwin
et
n1
llll'.
(l'J7H)
a vjtarnin D-dependent calcium binding prote in in r{lt pa!
130
{II.
t
id
1 1 1 1
gland using the Ghelex method,
Tenenhouse & Glijer (unpub1ished) were
unah le to reproduce these findings using the same technique as well as a electrophoretic assay that readiIy detected these proteins
45 Ca 2+
kidney,
gut
and
cerebellum.
Moreover,
M.
Thomasett
in
(personal
communication) could not find evidence of either calbindin-D 9k or calbindin-D 28k in parotid gland using monoclonal antibodies specif':'" for
)
lhe,Sc' proteins.
J
1,25 (OH)2D3 receptors in parotid acinar cells in strong support of the
Despi te
these
uncertainties,
l
considered
the presence
of
original hypothesis that this gland was a target organ for vi tamin D.
)
J
4.2.
Pilocarpine-Stimulated Secretion of Parotid Saliva by Gl and G2 Ca-/D- Rats In Vivo
j .1
The effect of vitamin D deprivation on parotid gland function was investigated next.
J J
1
10-) al
and their offspring (G2 Ca- /D-) were raised on a
least 7 weeks.
(h'ficient
by
1
this
measurable
arter
l"e found to have severely reduced maximal f10w rates compared to thosC' of rats
(Fig.
18).
Secretion
of the
fluid
phnse of
parotid saliva appeared to be selectively impaired, as normal amounts of salivary amylase were secreted by the same anima1s (Fig.
23). TrelltlllC'nt rat~,
with D3 comp1etely restored normal salivary flow rates in G2 Ca-/D(Fig.
18).
In contrast,
1,25 (OH) 203 in blood, pilocarpine respect
(Fig.
except
GI Ca-/D-
exhibi ted a
l,25(OH)2D3
rats,
which
still
had dctectnblp
normal parotid secretory response to
17). Since the
two groups were similar
status,
these
findings suggested
in pvpry that liH'
abnorma1ity in parotid gland function was a direct result of the BbsPl1cf' and hyperparathyroidism.
not
secondary
ta
hypocalcemia
It was concluded at that time that l, 25(OH) 2D)
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